Human leukotriene C4 synthase (LTC4S) is an integral membrane protein of interest as a target for the treatment of bronchial asthma and pulmonary fibrosis. LTC4S is highly specific for its substrate leukotriene (LT) A4, which it conjugates with reduced glutathione to form LTC4, responsible for the characteristic symptoms of asthma including bronchoconstriction, mucus hypersecretion and edema of the airways. To gain an understanding of the molecular mechanism and the consequences of the specificity for treatment as well as membrane protein structures in general, we propose to study human LTC4S by electron cryo-microscopy (cryo-EM), specifically electron crystallography. Our Preliminary Studies show that human LTC4S can be overexpressed and purified to an extent and in a quantity, which allows us to reproducibly induce two-dimensional (2D) crystallization. The resulting crystals are large and highly ordered, and projection data extending to better than 4E resolution visualizes four transmembrane 1-helices. Based on previous site-directed mutagenesis studies, we hypothesize that Arg-51 and Tyr-93 are involved in catalysis while Ile-27, Val-35, Val-49, Arg-51, Ala-52, Asn-55, Tyr-59, Tyr-93, Tyr-97, and Ala- 112 form the binding site for LTA4 and glutathione. Structural data would provide important information regarding the understanding of the arrangement and role of these residues and provide a basis for further functional studies by both biochemical and structural means. In Aim 1 of this proposal we will determine an atomic structure of human LTC4S by collecting and analyzing data of tilted and untilted 2D crystals by cryo-EM. The structure would allow us conclusions about the location and residues forming the active site. In Aim 2 we will crystallize various mutants of human LTC4S and carry out inhibition and activation studies to understand the detailed molecular mechanism and the high specificity of the enzyme. This information might eventually be used to explore LTC4S specific inhibitors.Project Narrative The atomic model of human leukotriene C4 synthase (LTC4S) and an insight into its reaction mechanism might lead to a better understanding and treatment of a number of inflammatory diseases, including asthma, allergic rhinitis, and pulmonary fibrosis. In particular, LTC4S specific inhibitors could be explored once the structure is available.